The availability of a large number of biological reagents, such as hundreds of thousands of cloned DNA sequences, numerous antibodies and recombinant proteins, millions of compounds obtained through combinatory chemical synthesis, has promoted the development of technologies that make use of these reagents in biological research, clinical diagnostics and drug development. Special position-addressable arrays of biological reagents have been designed, in which each of the reagents is placed at a pre-defined position so that it can be identified later by the position. For example, in a DNA array, a large number of cDNA or oligos are immobilized, each at a pre-defined position and can be identified later by that position. DNA arrays are used in large-scale hybridization assays for applications such as monitoring gene expressions (Schena et al., 1995, Science 270:467-470; DeRisi et al., 1996, Nature Genetics 14:457-460). Arrays of DNA clones in expression vectors are also used to express their encoded proteins in mammalian cells (Ziauddin and Sabatini, 2001, Nature 411, 107-110).
In a common type of protein array (capture protein array), many proteins are immobilized on a support, each at a predefined position so that every protein can be identified subsequently by its unique position. Capture protein arrays are used to capture ligands onto the array support for subsequent analysis. Two types of capture protein arrays are widely used: antibody arrays and recombinant protein arrays, which contain a plurality of antibodies and recombinant proteins, respectively. Antibody arrays are particularly useful in revealing protein expressions and activities: it is possible to use them to study the properties of a large number of cellular proteins in a single assay. Antibody arrays have been applied in studying in vivo protein-protein interactions, protein posttranslational modifications and protein expression patterns (U.S. Pat. No. 6,197,599).
In another type of protein arrays, dissociable protein arrays, proteins are immobilized on a support in such a way that the immobilized proteins can dissociate from the array support when placed in contact with their interacting ligands that are immobilized on another support (Wang, “Immunostaining with dissociable antibody microarrays”. Proteomics 4, 20-26. 2004). Dissociable protein arrays are used to deliver a plurality of reagents to their binding ligands in a position-addressable manner. They have been used in detecting protein expressions and subcellular localizations (Song et al. 2008 “Protein Expression Profiling of Breast Cancer Cells by Dissociable Antibody Microarray (DAMA) Staining”. Molecular & Cellular Proteomics 7:163-169. Fu et al. 2010 Protein Subcellular Localization Profiling of Breast Cancer Cells by Dissociable Antibody MicroArray (DAMA) Staining. Proteomics. 10(8):1536-44).
In addition, arrays of cells, tissues, lipids, polymers, drugs and other chemical substances can be fabricated for large scale screening assays in medical diagnostics, drug discovery, molecular biology, immunology and toxicology (Kononen, et al., Nature Medicine, 4:844-7, 1998).
Proteins are the major component of cells and they play important roles in various cellular processes. The entire human genome contains 20,000 to 25,000 protein-encoding genes. Although a given cell may contain the DNA encoding all the proteins, it usually only expresses a fraction of them. A cell line usually expresses about 10,000 proteins and a tissue may express an even higher number of proteins. The protein expression pattern of a cell determines its shape and function; and abnormal protein expressions cause cells to malfunction, resulting in diseases. Therefore, one major task of proteomics is to identify the protein expression patterns in a given source.
A protein with an identical primary amino acid sequence may be present in different forms in the cells largely due to posttranslational modifications. Since in many cases only special posttranslationally modified proteins are activated and directly involved in a cellular process, the detection of these activated proteins in the cells can provide valuable information on that cellular process. There are many types of protein posttranslational modifications including phosphorylation, glycosylation, and ubiquitination. And they play important roles in regulating protein activities. Phosphorylation in either serine, threonine or tyrosine residues is an important mechanism in signal transduction. Aberrant protein phosphorylation contributes to many human diseases. Among the methods of detecting protein phosphorylations, metabolic labeling of cells with radioisotopes and immuno-detection with antibodies against phosphoproteins are most commonly used. However, these methods are usually only applicable to the analysis of one or a few proteins at a time. Although antibodies specific for phosphorylated amino acids, such as PY20 and 4G10, can reveal multiple phosphorylated proteins, they alone are unable to identify individual phosphorylated proteins. New methods for simultaneously detecting the presence of multiple phosphorylated proteins or other modified proteins are highly desirable for signal transduction studies and clinical diagnosis.
Quantification of protein expressions has applications in a variety of fields including biomedical research, disease diagnosis, identification of therapeutic targets, and profiling cellular responses to toxins and pharmaceuticals. In basic biomedical research, it is usually desirable to know what proteins are expressed in specific cells or under specific conditions. And by comparing the protein expression profiles between different cell types, it is possible to identify those proteins whose expressions and activations characterize a particular cell type. In many signal transduction pathways, certain proteins are specifically activated; and the detection of these active proteins, e.g., phosphorylated proteins, may provide important information on the activations of specific signal transduction pathways.
Many diseases alter protein expressions and in many cases abnormal protein expressions are the causes of the diseases. Therefore, determination of protein expression profiles and comparison of the expression profiles between normal and abnormal biological samples are useful for understanding disease mechanisms. Detecting proteins is also useful in clinical diagnostics. For example, examination of the presence of several viral proteins instead of just one in a blood sample is a more reliable diagnostic method for viral infections. Profiling proteins will be invaluable in distinguishing normal cells from early-stage cancers and also from malignant, metastatic cancer cells that are the real killers. In addition, proteins are the targets of most drugs, and protein expression profiling is useful in key areas of drug development, such as in drug target selection, toxicology and the identification of surrogate markers of drug response.
It has long been the goal of molecular biologists to develop technologies that can quantify, in a reliable and reproducible manner, the expression level of every individual protein and the different forms of each protein in a biological sample. However, this has turned out to be extremely difficult to achieve. Traditionally, the expression of one or a small number of proteins can be detected by immunological methods, such as western blotting and Enzyme-Linked Immunosorbent Assay (ELISA).
Immunochemical staining is a versatile technique in determining both the presence and localization of an antigen (Harlow and Lane, Antibodies, a laboratory manual, Cold Spring Harbor Press, 1988). Two-dimensional gel electrophoresis can be used to analyze the proteins expressed in a sample. However, it requires complicated procedures and it is necessary to determine the identities of the proteins displayed on the two-dimensional gel, which is difficult to achieve for most proteins. Recently, protein arrays are applied in studying protein expression patterns. In one strategy (U.S. Pat. No. 6,197,599; Haab, et al., Genome Biol. 2, research 0004.1-0004.13, 2001), an antibody array is incubated with a protein sample and after incubation and washing, proteins specifically bound to their respective antibodies on the array are detected. The most challenging problem of the current protein array technology is low specificity. The problem is due primarily to the so-called “non-specific” binding of capture and detection reagents (e.g. antibodies). “Cross-talk” between those reagents creates false signals in protein array methods.